Abstract Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the p110 γ subunit playing a key role in immune signalling. PI3K γ is a key factor in inflammatory diseases, and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the c-terminus can inactivate or activate enzyme activity. Screening of small molecule inhibitors using HDX-MS revealed that activation loop binding inhibitors induce allosteric conformational changes that mimic those seen for the R1021C mutant. Structural analysis of clinically advanced PI3K inhibitors revealed novel binding pockets that can be exploited for further therapeutic development. Overall this work provides unique insight into the regulatory mechanisms that control PI3K γ kinase activity, and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveals a path to selective inhibitors